SAR by WaterLOGSY?

Among ligand-based NMR methods, WaterLOGSY is nearly as popular as STD NMR. Normally the information obtained is limited: does a given small molecule bind to a protein or not? In a new paper in J. Enzyme Inhib. Med. Chem., Isabelle Krimm and collaborators at the Université de Lyon and University of York try to wring more data from this common experiment.

In WaterLOGSY, magnetization is transferred from water, to protein, and then to bound ligand. This can happen through multiple mechanisms, and even talented NMR spectroscopists have told me they have trouble understanding exactly what is going on. In short, the WaterLOGSY spectra of molecules bound to proteins show a change in sign compared to molecules that don’t bind. Examining ligands in the presence and absence of protein can thus provide evidence for whether a ligand binds.

The researchers go beyond this simple qualitative approach and look at changes in peaks corresponding to specific hydrogen atoms in each ligand. They define a “WLOGSY factor,” which shows an inverse correlation to solvent exposure. In other words, a smaller WLOGSY factor means that a given hydrogen atom in a ligand is more exposed to water, and thus less exposed to protein. If all the hydrogen atoms in a bound ligand have the same WLOGSY factor, this suggests either multiple binding modes, or that the ligand is completely enclosed by the protein. If, on the other hand, different hydrogen atoms in a bound ligand have different WLOGSY factors, this could provide information on the binding mode. This analysis is conceptually similar to the STD epitope mapping the Krimm lab described several years ago, and STD experiments were also run on the proteins here for comparison.

To validate the approach, the researchers tested six proteins (with molecular weights ranging from 22 to 180 kDa) for which fragment ligands had been previously identified with affinities from 50 µM to worse than 1 mM. Screens were done using 400 µM fragment and 5 to 20 µM protein. (NMR aficionados, please see the paper for details on the effects of mixing times and ligand exchangeable protons.)

The results look pretty impressive: for PRDX5, HSP90, Bcl-x_L, Mcl-1, and glycogen phosphorylase, the ligand hydrogen atoms previously shown to be solvent exposed from crystallographic or two-dimensional NMR structures do in fact show reduced WLOGSY factors. In the case of human serum albumin, a ligand showed uniform WLOGSY factors, suggesting multiple binding modes, as expected given the multiple promiscuous binding sites on this protein.

To a non-NMR spectroscopist such as myself, this seems like a useful approach for obtaining binding information in the absence of crystallographic data. It also seems easier to run than the LOGSY titration we highlighted a couple years ago. But the first word of this blog is “Practical.” We recently discussed work demonstrating that STD NMR data is perhaps not as easily interpretable as many assume. Have you tried anything like this yourself, and if so how well does it actually work?

Fragments vs the PWWP1 domain of NSD3: a chemical probe

Epigenetics is a topic we’ve covered frequently at Practical Fragments. Much attention has been focused on bromodomains, which recognize acetylated lysine residues. However, lysine side chains are also methylated to affect gene expression. The PWWP1 domain of the protein NSD3 (NSD3-PWWP1) recognizes these modified lysines. This protein is amplified in several tumour types, and so makes an intriguing cancer target. At the CHI DDC conference last year Jark Böttcher presented how Boehringer Ingelheim and a large multinational group of collaborators developed a chemical probe for NSD3. The story now appears in Nat. Chem. Biol. (and see here for a fun animated short set to music).

The researchers started by screening a library of 1899 fragments against NSD3-PWWP1. STD NMR (at 0.25 mM of each fragment, in pools of four) as well as differential scanning fluorimetry (at 0.5 mM of each fragment) resulted in 285 and 20 hits, respectively. Two-dimensional NMR was used to confirm hits. Interestingly, only three fragments were identified from both STD-NMR and DSF, and these did not confirm – a cautionary reminder that screening orthogonal methods is not necessarily the best path.

Fortunately, 15 fragments not only confirmed, but also caused the same changes to the 2D-NMR spectra as a histone-derived peptide containing a dimethyl-lysine residue, suggesting that the fragments bind at the recognition site for modified lysines. Those fragments with dissociation constants better than 2 mM were pursued crystallographically, and some of the successes included compound 4. This molecule was used in a virtual SAR-by-catalog screen of internal compounds. Of the 601 fragments experimentally tested, compound 8 was the most potent. Crystallography confirmed that the compound binds in the expected site, and further structure-based design ultimately led to BI-9321.

BI-9321 was put through a battery of tests. Affinity was confirmed in biochemical, SPR, and ITC assays, and crystallography revealed the binding mode to be similar to the initial fragment. BI-9321 was selective for NSD3-PWWP1 when tested against 14 other PWWP domains, and showed no activity against 35 protein methyltransferases, 31 kinases, and 48 bromodomains. Solubility, in vitro metabolic stability, permeability, and plasma protein binding all look good.

Multiple assays also demonstrated selective target engagement in cells at a concentration of around 1 µM. BI-9321 showed downregulation of MYC mRNA levels, though the effect was both modest and transient. Antiproliferative activity was also observed in cells, and the effects were synergistic with a bromodomain inhibitor. Moreover, these effects were only seen in NSD3-dependent cells, suggesting that the activity is on-target and that the compound is not generally cytotoxic.

All of this makes BI-9321 an attractive chemical probe, at least for cell-based assays. More work will need to be done to improve potency and further understand the biology. Laudably, to this end, the researchers have made the molecule publicly available.

Fragments vs viral protein EBNA1

Epstein-Barr virus (EBV) infects more than 90% of adults. In most cases it remains latent, but even then it expresses genes that cause cellular proliferation, which can lead to cancer. In fact, up to 2% of human cancers are caused by the virus. In a recent paper in Sci. Transl. Med., Troy Messick, Paul Lieberman (both at the Wistar Institute) and a large group of collaborators (including Teddy Zartler) take aim at this pathogen.

The researchers focused on the viral protein EBNA1, a DNA-binding protein essential for viral replication as well as host cell survival. They started with a virtual screen of 1500 fragments from Maybridge, and then did fragment soaking of the top 100 hits. Happily, this resulted in structures of 20 fragments in four separate sites on the protein. Less happily for modelers, none of the fragments bound as predicted – more grist for the “crystallography first” argument.

A dozen fragments bound in a deep hydrophobic pocket, and most of them contained an acidic moiety that made hydrogen bond contacts to conserved threonine and asparagine residues that normally contact the DNA backbone. Merging two of these fragments led to VK-0497, which disrupts binding of EBNA1 to DNA at sub-micromolar concentrations. Crystallography confirmed that it bound as expected. The molecule contains a potentially unstable pyrrole, and replacing this with an indole and growing led to molecules such as VK-1248, with high nanomolar activity in the DNA-binding assay. Additional biophysical techniques including SPR, ITC, and 2-dimensional (HSQC) NMR confirmed binding for this and related compounds.

The carboxylic acid moiety likely reduces permeability across cell membranes, and indeed the compounds showed no activity in cells. However, methyl esters were found to be rapidly cleaved by intracellular esterases, and these prodrugs were tested in a variety of assays.

The prodrugs inhibited proliferation of EBV-positive human cells but had no effect on non-infected cells. The prodrugs also reduced expression of both viral and host proteins. More importantly, they inhibited tumor growth in xenograft models using four different cancer cell lines, two of which were patient-derived. Prolonged dosing over as long as eight weeks showed a sustained effect, which is reassuring in terms of drug resistance. Finally, the molecules could effectively be combined with existing drugs or radiation.

There is still much work to be done, not just in terms of potency but also further pharmacokinetics, pharmacology and toxicology. And as the researchers acknowledge, xenograft models are regrettably poor surrogates for humans. Still, this is an interesting approach, and hopefully further work will be done on this series, or at least the target.

Stabilizing apolipoprotein E4 with fragments

Among fragment-derived drugs that have entered the clinic, BACE1 inhibitors are well-represented. Sadly, multiple drugs targeting this protein have failed to show efficacy against Alzheimer’s disease. That said, every drug that has been thrown at Alzheimer’s has failed to slow the disease, so perhaps we need to think more boldly. An example was published recently in J. Med. Chem. by Andrew Petros, Eric Mohler, and colleagues at AbbVie.

The researchers were interested in apolipoprotein E4 (apoE4), one of three isoforms found in humans. Folks who have two apoE4 alleles are at increased risk for Alzheimer’s, suggesting that the protein might make a good drug target. Unfortunately, although it is known to be a lipid carrier, its precise function is unclear. What is known is that apoE4 is less stable to thermal denaturation than apoE2 or apoE3, so the team set out to find molecules that would stabilize the protein. This being AbbVie, they used two-dimensional NMR to find fragments.

The methyl groups of all the isoleucine, leucine, methionine, and valine residues in apoE4 were ¹³C labeled, and the researchers looked for changes in the ¹³C-HSQC spectra upon addition of fragments; just over 4000 were screened in pools of 12, each at 1 mM. Of the dozen or so hits, compound 1 was among the best.

NMR titration studies revealed an affinity just under 1 mM, while SPR suggested slightly stronger binding. As hoped, compound 1 raised the melting temperature of apoE4. Adding the fragment also altered the kinetics of liposome breakdown, causing the protein to behave more like apoE2 and apoE3. Although this assay isn’t necessarily physiologically relevant, the reasoning is that causing apoE4 to behave more like the other isoforms may be useful.

A crystal structure of compound 1 bound to apoE4 revealed the fragment to be binding in a small pocket, and growing led to compound 2, with a slightly improved affinity. Introduction of polar substituents to interact with a nearby aspartic acid side chain led to compound 8, with low micromolar affinity (assessed by NMR). This molecule also stabilized apoE4 with respect to thermal denaturation.

As noted above, it is not entirely clear why apoE4 is associated with Alzheimer’s, but researchers had previously found that overexpression in a neuronal cell line caused release of the inflammatory cytokines IL-6 and IL-8. When human induced pluripotent stem cell (iPSC)-derived astrocytes carrying two copies of apoE4 were treated with compound 8, release of IL-6 and IL-8 cytokines was reduced to levels similar to those from iPSC-derived astrocytes carrying two copies of apoE3. The compound also showed no toxicity, even at relatively high concentrations (100 µM).

There is still a tremendous amount to do: affinity needs to be improved considerably, and permeability is also mentioned as an issue. Moreover, the highly polar nature of compound 8 will likely make transport across the blood-brain barrier challenging. Optimizing activity against a target whose function is poorly understood will present a host of problems. But if it were easy, Alzheimer’s disease would not be the scourge that it is. Practical Fragments salutes thinking outside the box, and wishes those involved the best of luck.

Fragment events in 2019 and 2020

We're already halfway through 2019, but there are still some excellent upcoming events, and 2020 is taking shape, so start planning your calendar!

2019
September 1-4: BrazMedChem2019 will be held in the Brazilian holiday destination of Pirinopolis, and will include a section on FBLD.

September 17-19: CHI's Seventeenth Annual Discovery on Target takes place in Boston. Multiple biological targets are covered, and there are also more general talks on a variety of topics of interest to readers, including two sessions on fragments. You can read my impressions of last year's event here.

November 12-15: FBDD Down Under 2019 will take place in beautiful Melbourne. This is the third major FBDD event in Australia, and given the success of the first, I expect it to be excellent.

November 13-15: Although not exclusively fragment-focused, the Seventh NovAliX Conference on Biophysics in Drug Discovery will have several relevant talks, and will be held in the lovely city of Kyoto. You can read my impressions of the 2018 Boston event here, the 2017 Strasbourg event here, and Teddy's impressions of the 2013 event here, here, and here.

2020
April 13-17: CHI’s Fifteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of the 2019 meeting here, the 2018 meeting here, the 2017 meeting here, the 2016 meeting here; the 2015 meeting here, here, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here.

September 20-23: FBLD 2020 will be held for the first time in the original Cambridge (UK). This will mark the eighth in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2018, FBLD 2016, FBLD 2014,  FBLD 2012, FBLD 2010, and FBLD 2009.

December 15-20: The second Pacifichem Symposium devoted to fragments will be held in Honolulu, Hawaii. The Pacifichem conferences are held every 5 years and are designed to bring together scientists from Pacific Rim countries including Australia, Canada, China, Japan, Korea, New Zealand, and the US. Here are my impressions of the 2015 event.

Know of anything else? Add it to the comments or let us know!